Regulating TXNIP/NLRP3 And TXNIP/ChREBP Pathways By Salvianolic Acid A Alleviates Non-alcoholic Liver Injury In Rats

Background:Non-alcoholic fatty liver disease(NAFLD)has a worldwide distribution and is now considered the leading cause of chronic liver disease.Thioredoxin-interacting protein(TXNIP,also known as TBP-2,TRX-binding protein-2)is the endogenous inhibitor and regulator of the thioredoxin(TRX)system,which plays an important role in diverse cellular processes,including the regulation of celluar redox balance,inflammation,lipid metabolism and apoptosis.NLRP3 inflammasome has been reported to mediate the sterile inflammation and lead to tissue damage due to increasing pro-inflammatory cytokines secretion,including IL-1? and IL-18.Furthermore,recent literatures suggested TXNIP as a critical signaling molecule linking oxidative stress to NLRP3 inflammasome activation,and that TXNIP/NLRP3 activation may be involved in the pathogenesis of NAFLD.Carbohydrate response element-binding protein(ChREBP),a transcriptional activator of glycolytic and lipogenic genes,has emerged as a key determinant of the control of hepatic de novo fatty acid synthesis under physiological conditions as well as in the context of NAFLD.Moreover,recent studies have showed that TXNIP modulates ChREBP activity in INS-1 cells and in mouse islets,resulting in a regulation of other ChREBP target genes playing important roles in glucose and lipid metabolism.Taken together,TXNIP may be a regulator of NLRP3 inflammasome activation and ChREBP activity.Salvianolic acid A(SalA),one of the most efficacious polyphenol compounds extracted from the Radix Salvia miltiorrhiza(Danshen),has been shown to possess lots of potential pharmacological activities.The effect of SalA on NAFLD has not been reported.Besides,it becomes recognized that some phenolic compounds may conferred a protective effects by targeting TXNIP.However,it remains unknown whether SalA-mediated protective effects involve TXNIP/NLRP3 and TXNIP/ChREBP pathways.Objective:To investigate the involvement of TXNIP-NLRP3 and TXNIP-ChREBP pathways in NAFLD.To explore the protective effect of SalA,as well as the exact molecular mechanisms,in HFD-induced NAFLD.Methods:1.Fifty healthy male Sprague-Dawley(SD)rats weighting 180-220g,were randomly separated into five groups:1)control group,2)control+SalA(16 mg/kg/d)group,3)high-fat diet(HFD)group,4)HFD+SalA(8 mg/kg/d)group,and 5)HFD+SalA(16 mg/kg/d)group.The rats were housed in individual microisolator cages with free access to sterile water and an irradiated standard diet or high-fat diet(66%base forage,2%cholesterol,7%lard,8.3%yolk and 16.7%sucrose)in a special pathogen-free facility.Rats underwent the intragastric administration of SalA(8 and 16 mg/kg/d)or the same volume of normal saline for 10 weeks.After exposure to the high-fat diet for 10 weeks,all of the animals were euthanized.The blood and liver samples were harvested for analysis.The serum levels of Alanine Aminotransferase(ALT)?Aspartate Aminotransferase(AST)?Triglycerides(TG)?Total Cholesterol(TC)?Non-esterified fatty acid(NEFA)?Tumor necrosis a(TNF-?)?Interleukin-6(IL-6)and TG?TC?NEFA?H2O2?Malondialdehyde(MDA)?Superoxide dismutase(SOD)activity in the liver were performed according to manufacturer's instructions.qRT-PCR was used to determine the expression of TXNIP and NLRP3 in the liver.Western blot analysis was used to test the expressions of TXNIP.NLRP3?ASC?Caspase-1?IL-1??ChREBP and FAS.2.(1)The HepG2 cells were divided into five groups:the control group,the palmitic acid(PA)group and the SalA pretreatment groups(0.2?M,20?M,20?M).The PA group and the SalA pretreatment groups were treated with PA(500?M)for 24 hours.MTT method was used to detect cell survival rate.(2)The HepG2 cells were divided into three groups:the control group,PA group and the PA+SalA(20?M)groups.The intracellular lipid droplets was detected by Nile red stains and the levels of TNF-a and IL-6 in the culture medium were measured using commercially available ELISA kits.Levels of ROS and H2O2 were measured according to manufacturer's instructions.(3)The HepG2 cells were divided into four groups:the control group,the PA group,the PA+SalA(20?M)group and the PA+Resveratrol(RES)(10?M)group.Western blot analysis was used to test the expressions of TXNIP?NLRP3?ASC?Caspase-1 and IL-1?.(4)The HepG2 cells were divided into four groups:negative control group,negative control+SalA(20?M)group,siRNA TXNIP group and siRNA TXNIP+SalA(20?M)group.The protein expression of TXNIP and NLRP3 was detected by Western Blotting.(5)The HepG2 cells were divided into five groups:negative control group,negative control+PA group,negative control+PA+SalA(20?M)group,siRNA TXNIP+PA group and siRNA TXNIP+PA+SalA(20?M)group.The protein expression of TXNIP?NLRP3?ASC?Caspase-1?IL-1? and FAS in the whole cell lysate and ChREBP both in nucleus and cytoplasm was detected by Western Blotting.The levels of TNF-a and IL-6 in the culture medium were measured using commercially available ELISA kits.(6)The HepG2 cells were divided into five groups:the control group,the PA group,the PA+SalA(20?M)group,the PA+BAY 11-7082(15?M)group and the PA+SalA(20?M)+BAY 11-7082(15?M)group.The protein expression of TXNIP?NLRP3 and IL-1? was detected by Western Blotting.Results:In vivo,SalA treatment significantly attenuated HFD-induced obesity,remarkably decreased the serum levels of ALT and AST,as well as the liver and serum levels of TG,TC and NEFA.Moreover,SalA treatment ameliorated HFD-induced hepatic inflammation and oxidative stress by decreasing hepatotoxic cytokines levels such as IL-6 and TNF-a,suppressing the overproduction of reactive oxygen species(ROS)and MDA,preventing the decreased expression of SOD.Importantly,SalA reversed the HFD-or PA-induced activation of NLRP3 inflammasome,nuclear translocation of ChREBP and up-regulation of FAS,accompanied by down-regulation of TXNIP.In addition,TXNIP siRNA abrogated the effects of SalA on PA-induced NLRP3 inflammasome activation,IL-1? overexpression and secretion of pro-inflammation cytokines,as well as nuclear translocation of ChREBP and up-regulation of FAS.Moreover,pharmacological inhibition of NLRP3 inflammasome by BAY 11-7082(one of NLRP3 inflammasome specific inhibitors)abolished PA-induced NLRP3 and IL-1?overexpression,but had no effects on TXNIP expression.Conclusion:TXNIP-NLRP3 and TXNIP-ChREBP pathways are crucial during HFD-induced NAFLD,and SalA confers protection against NAFLD,at least in part through regulating TXNIP-NLRP3 and TXNIP-ChREBP pathways.